Methodology for the preparation and analysis of N-tris(2,4,6-trimethoxyphenyl)phosphonium-acetyl (TMPP-Ac) derivatives of peptides has been developed in this laboratory. Most often we analyze the TMPP-Ac derivatives of peptides by FAB with subsequent CAD or by MALDI with subsequent PSD to obtain sequence information. We are investigating the analysis of the TMPP-Ac derivatives by ESI with in-source fragmentation to obtain sequence information. Our preliminary experiments show that in-source fragmentation of the TMPP-Ac derivatized peptides is sensitive to different parameters such as cone voltage, capillary voltage, nebulizing gas flow rate, etc. The developing methodology is being extended to TMPP-Ac derivatization of peptide fragments from tryptic digests of proteins with subsequent analysis by LC-MS using electrospray.